The Effect of Calcium and Glucose Concentration on Corneal Epithelial Cell Lines Differentiation, Proliferation, and Focal Adhesion Expression

Abstract

It is known that culture media composition can affect cell behavior, morphology, and gene expression. However, in the case of corneal epithelial cells, the combined role of calcium and glucose concentration in media has not previously been examined. In this study, a human immortalized corneal epithelial cell line was used to examine the effect of glucose and calcium concentrations on these cells. Cell metabolic activity, cell growth curve analysis, and relative gene and protein expression of proliferative marker extracellular related kinase (ERK) were used to study proliferation. Corneal epithelial stem cell marker NP63 and mature epithelial marker cytokeratin 3 (CK3) were analyzed by using reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry. Focal adhesions were examined by using immunocytochemistry. Cells cultured in both low-glucose, high-calcium (LG-HC) media and high-glucose, low-calcium (HG-LC) media showed similar results in both RT-PCR and immunocytochemistry analysis. NP63 expression was significantly lower and CK3 expression was higher in these groups compared with cells cultured in commercial media. NP63 and CK3 expression was also analyzed by using immunocytochemistry, which confirmed these findings. The high-glucose, high-calcium-fed cells showed the lowest expression of all markers and no gene expression of CK3. This was deemed the most unsuitable media formulation for this cell line. Focal adhesion expression was the lowest in the high-calcium, high-glucose-fed cells, with the most even distribution of this among the commercial media group. Overall, this study showed that varying glucose and calcium concentrations can have significant effects on differentiation, proliferation, focal adhesions, and metabolic activity of this cell line. It seems that an LG-HC and HG-LC formulation were interchangeable with similar proliferative and differentiation effects.

Keywords: calcium; cornea; epithelium; glucose.

FIG. 1.

Cell growth curve of hTCEpi cells grown in different media formulations over 7 days. Cells grown in the commercial media displayed a significantly higher cell number over 7 days compared with all other groups. The LG-HC group had a significantly higher rate of growth compared with the HG-HC and HG-LC group. Data are presented as the mean ± SD. HG-HC, high glucose, high calcium; HG-LC, high glucose, low calcium; LG-HC, low glucose, high calcium; SD, standard deviation.

FIG. 2.

Metabolic activity of hTCEpi cells after 7 days in culture. Cell metabolic activity was significantly decreased in all groups compared with commercial media-cultured cells. Data are presented as the mean ± SD, significance calculated via one-way ANOVA with Post-Tukey test, n = 3, ****p ≤ 0.0001. ANOVA, analysis of variance.

FIG. 3.

RT-PCR to determine relative expression of ERK after 7 days in culture. HG-HC-fed cells expressed ERK significantly lower than the commercial media-cultured cells and the LG-HC group. Data are presented as the mean ± SD, significance calculated via one-way ANOVA with Post-Tukey test, n = 3, *p ≤ 0.005, **p ≤ 0.01. ERK, extracellular-related kinase; PCR, polymerase chain reaction.

FIG. 4.

RT-PCR to determine relative expression of NP63 after 7 days in culture. HG-LC and HG-HC groups had the most significant decrease in NP63 expression compared with the commercial media-cultured cells followed by LG-HC. Between groups, the HG-HC group and the HG-LC expressed NP63 significantly lower than the LG-HC group. Data are presented as the mean ± SD, significance calculated via one-way ANOVA with Post-Tukey test, n = 3, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

FIG. 5.

RT-PCR to determine relative expression of CK3 after 7 days in culture. Cells fed in LG-HC and HG-LC media expressed CK3 higher than the commercial media-cultured cells, and CK3 was not detected (nd) in the HG-HC group. No significance was observed between groups or compared with control. Data are presented as the mean ± SD, n = 3. CK3, cytokeratin 3.

FIG. 6.

Western blot and densitometry analysis of a corneal epithelial cell line cultured in different media formulations after 7 days. No significant differences between groups were observed of pERK expression. Data are presented as the mean ± SD, n = 3.

FIG. 7.

Immunocytochemistry analysis of a corneal epithelial cell line cultured in different media formulations after 7 days. Top row (A–D) cells stained in green for NP63 (green); second row (E–H) cells stained in green for CK3; and bottom row (I–L) cell stained in green for vinculin. All cells were counterstained for f-actin (red) and DAPI (blue). First column (A, E, I) shows cells cultured in commercial media; second column (B, F, J) shows cells cultured in LG-HC; third column (C, G, K) shows cells cultured in HG-HC; and fourth column (D, H, L) shows cells cultured in HG-LC (Scale bar = 10 μm). DAPI, 4′,6-diamidino-2-phenylindole.